7,8 Although RNA is easily and successfully isolated from most cells and tissues, intact RNA extraction from the pancreas is difficult due to the high level of its ribonucleases (RNases). Despite the improvement in several approaches, including rapid removal of pancreatic LEE011 cell line tissue from the abdominal cavity and homogenization at cold temperatures to inhibit RNases, the isolation of intact, high-quality RNA from this tissue remains challenging because of the complexity and indefinite reproducibility of the above mentioned techniques.9-15 We aimed to design a simple, fast, and cost-effective method for complete RNA extraction that utilized the least
amount of pancreatic Inhibitors,research,lifescience,medical tissue. We compared different protocols of
RNA extraction and optimized the most feasible extraction method by which the highest quality RNA could be qualitatively obtained. Materials and Methods In the current study, pancreatic tissues were taken from 30 rats and divided into several pieces (20-30 mg) Inhibitors,research,lifescience,medical for use in the following methods. In the first method, these small pieces of pancreatic tissue from 30 rats were placed into two microtubes. Inhibitors,research,lifescience,medical The first tube contained 1 ml RNX-plus solution (Cinnagen, Tehran, Iran) and the second tube contained 1 ml TriPure isolation reagent (Roche Applied Science, Germany). Both solutions contained guanidinium thiocyanate which inhibits RNase. Subsequently, both tubes were snap-frozen in liquid-nitrogen for inhibition of RNase activity after which the integrity of RNA was evaluated with denaturing agarose gel electrophoresis (figures 1 and and22). Figure 1 Evaluation of total RNA integrity isolated Inhibitors,research,lifescience,medical from three snap-frozen pancreatic tissues using RNX-plus. Lane 1 shows the quality of RNA extracted from the liver as the control. Lanes 2-4 represent the quality of 28S/18S rRNA bands in total RNA extracted
… Figure 2 Evaluation of total RNA integrity isolated from three snap-frozen pancreatic tissues using TriPure solution. Lane 1 shows the quality of RNA extracted Inhibitors,research,lifescience,medical from liver tissue as the control. Lanes 2-4 represent Etomidate the quality of 28S/18S rRNA bands in total RNA … In the second method, pancreatic tissues were perfused with 1 ml RNA-later as the RNA stabilization reagent (Qiagen, USA) by an insulin syringe. Tissues were subsequently cut into small pieces with sterile scissors. The tubes that contained pancreatic tissue and RNA-later were processed for extraction by using the RNX-plus solution, TriPure, and RNeasy Micro Kits (Qiagen, USA) according to the manufacturers’ instructions after either 30 min, overnight in 4ºC, or following storage at -80ºC for one, three or seven days in order to compare the effect of preservation time on RNA integrity. In all conditions, the livers were removed from 30 rats and used as control tissue in a comparison of RNA quality between pancreatic and liver RNAs.